The binding of the bisquaternary muscarine receptor antagonist heptane-1,7-bis(dimethyl-3'-phthalimidopropyl)-ammonium bromide (C7/3-phth) was investigated at a number of cholinergic binding sites using (-)-[3H]nicotine, [3H]pirenzepine and (-)-[3H]quinuclidinyl benzilate ([3H]QNB) in both central and peripheral tissues. C7/3-phth displayed an affinity for muscarine M2 receptors in rat atria (70.1 nM) which was 1.6-fold greater than for putative M4 receptors in rabbit lung, and 4- to 5-fold greater than for M1 receptors in rat cerebral cortex. Its affinity for nicotine receptors in the cortex was low, being 808-fold lower than its affinity for the M2 receptor. Although the displacement of (-)-[3H]nicotine and [3H]pirenzepine binding in rat cortex by C7/3-phth was best described in terms of one-site modelling, low Hill coefficients were observed with C7/3-phth in displacement studies using [3H]QNB in this tissue. The possibility of allosteric interactions or multiple receptor subtype interactions is discussed.